Purple acid phosphatases (PAPs) catalyze the hydrolysis of a wide range of activated phosphoric acid mono- and di-esters and anhydrides (Klabunde et al., 1996). The PAP proteins are characterized by seven conserved amino acid residues (shown in bold face) in the five conserved motifs XDXX, XDXXY, GNH(D/E), XXXH, XHXH, which are involved in the coordination of the dimetal nuclear center (Fe3+-Me2+) in the active site (Li et al., 2002), where Me is a transition metal and Me2+ is mostly found to be Fe2+ in mammalian, and Zn2+, or Mn2+ in plants (Klabunde and Krebs, 1997; Schenk et al., 1999).
Purple acid phosphatases are distinguished from the other phosphatases by their characteristic purple color, which is caused by a charge transfer transition at 560 nm from a metal-coordinating tyrosine to the metal ligand Fe3+ (Klabunde and Krebs, 1997; Schenk et al., 2000). Different from the other acid phosphatases, PAPs are insensitive to inhibition by tartrate, so they are also known as tartrate-resistant acid phosphatases (TRAPs).
The biochemical properties of some plant PAPs have been characterized, firstly in red kidney bean, and later in soybean suspension cell, soybean seedlings, rice culture cells, spinach leaves, sweet potato tubers, tomato, yellow lupin seeds, medicago and Arabidopsis, etc. (Schenk et al., 1999). Plant PAPs are generally considered to mediate phosphorus acquisition and redistribution based on their ability to hydrolyze phosphate compounds (Cashikar et al., 1997; Bozzo et al., 2004; Lung et al., 2008). Regulation of some plant PAPs transcripts by external phosphate level in medium or soil, strongly suggest their involving in phosphate acquisition. For example, the transcription level of Medicago MtPAP1 in roots was increased under P stress, implicating a role in P acquisition or internal mobilization (Xiao et al., 2005; Xiao et al., 2006). Some plant PAPs could be secreted from root cells to extracellular environment, then hydrolyze various phosphate esters. Lung et al. purified a secreted PAP phosphatase from tobacco, which could hydrolyze broad substrates and help to alleviate P starvation (Lung et al., 2008). Certain plant PAPs can also hydrolyze phytate, a major storage compound of phosphorus in plants. Hegeman and Grabeu (2001) purified a novel PAPs (GmPhy) from the cotyledon of the germinating soybean seedlings. GmPhy was introduced into soybean tissue culture and was assayed to show phosphatase activity. Most recently, AtPAP15 and 23 in Arabidopsis sharing high sequence homology (73-52%) with this soybean PAP, were found to exhibit phytase activity (Zhu et al., 2005; Zhang et al., 2008).
Besides involvement in P acquisition, plant PAPs may perform some other physiological roles. For example, the PAPs AtACP5 (AtPAP17), SAP1, and SAP2 (del Pozo et al., 1999; Bozzo et al., 2002) display not only phosphatase but also peroxidase activity, suggesting their involvement in the removal of reactive oxygen compounds in plant organs. A pollen-specific PAP from Ester lily was suggested to function as an iron carrier in mature pollen (Kim and Gynheung, 1996). Other studies indicate that plant PAPs may also be involved in NaCl stress adaption or cell regeneration (Kaida, 2003; Liao et al., 2003).
In the Arabidopsis genome, twenty-nine potential PAP genes were identified based on sequence comparison. Twenty-four of these putative enzymes contain seven conserved amino-acids residues involved in metal binding. One (AtPAP13) lacked four of these seven residues, and the other four (AtPAP14, 16, 28 and 29) lacked either the first, the second, or both motifs of the five conserved motifs. Twenty-eight are actively transcribed in Arabidopsis (Zhu et al., 2005).
To date, relatively little is known about AtPAPs biochemical properties and physiological roles, though several members have been characterized (del Pozo et al., 1999). AtPAP17 (AtACP5) was first known to be induced by phosphorus starvation. The transcription of AtPAP17 was also responsive to ABA, salt stress (NaCl), oxidative stress (H2O2) and leaves senescence, according to GUS activity assay. No alteration in the expression of AtPAP17 was observed during the nitrogen or potassium starvation, and paraquat or salicylic acid. Like the other type 5 acid phosphatases, AtPAP17 displayed peroxidation activity, which may be involved in the metabolism of reactive oxygen species in stressed or senescent parts of plants.
Besides AtPAP17, several AtPAPs were found to be involved in phosphorus metabolism in Arabidopsis. Root secretion of AtPAP12 was induced by P stress, and its regulation was mainly at transcriptional level (Patel et al., 1998; Coello, 2002/11). AtPAP4, as well as AtPAP10, AtPAP11 and AtPAP12 were involved in phosphorus starvation response since their transcription levels increased during phosphate deprivation (Li et al., 2002; Wu et al., 2003). In contrast, AtPAP20, 21 and 22 were irrespective to P starvation and expressed constitutively in Pi sufficient or deficient condition. Fluorescent signals were detected in the cytoplasm via the baculovirus expression system, indicating that they may function in the cytoplasm (Li and Wang, 2003).
AtPAP26 was purified and characterized from Pi-starved Arabidopsis suspension cell culture (Veljanovski et al., 2006). It exists as a homodimer with 55 kDa glycosylated protein, showing wide substrate specificity with the highest activity against phosphoenolpyruvate (PEP) and polypeptide phosphate. AtPAP26 also displayed alkaline peroxidase activity with the probable roles in the metabolism of reactive oxygen species. Proteomic study suggested that it may be localized in vacuole, and involved in recycling Pi from intracellular P metabolites (Shimaoka et al., 2004).
PAPs can act on a wide range of substrates, but not all of them exhibit phytase activity. An enzyme assay involving the GST-AtPAP23 fusion protein revealed that AtPAP23 exhibits phytase activity. A GUS study showed that AtPAP23 is exclusively expressed in the flower of the Arabidopsis, and may play certain roles in flower development (Zhu et al., 2005). In a recent report, a recombinant AtPAP15 expressed and partial purified in E. coli and yeast was also found to exhibit phytase activity) (Zhang et al., 2008). It was proposed that AtPAP15 may be involved in ascorbic acid biosynthesis with the end product myo-inositol of phytate hydrolysis as the precursor of ascorbic acid synthesis.
As stated above, most of the functions of characterized plant PAPs are related to phosphorus metabolism. None of the functionally or biochemically characterized plant PAPs carry transmembrane motif, and none of them were shown to be associated with membrane. Furthermore, to date, no AtPAPs or any plant PAPs, have been showed to affect sugar signalling and carbon metabolism in plant.
The first report of transgenic expression of plant PAP in plant was reported in 2005 (Xiao et al., 2005). The PAP-phosphatase gene from Medicago (MtPHY1) was expressed in transgenic Arabidopsis, resulting in increased capacity of P acquisition from phytate in agar culture (Xiao et al., 2005). Nonetheless, the growth performance of the plants was not reported to be different under normal growth.